SUMMARY / ABSTRACT Tobacco use, and most-prominently cigarette smoking, is the leading cause of preventable death in the USA and across the world. Smoking behavior is driven by addiction to nicotine, which exerts its effects through nicotinic acetylcholine receptors (nAChR). Better understanding how nAChR function and expression is regulated is thus crucial. Prototoxins are an extensive family of proteins which serve as physiologically important regulators of multiple nAChR subtypes. However, the basis of prototoxins' nAChR subtype selectivities, their interaction sites, the mechanisms by which they alter nAChR function, and their roles within nicotine dependence pathways are largely undetermined. This application addresses these critical gaps in our knowledge. Our Preliminary Data indicate that the prototoxin lynx1 allosterically regulates multiple isoforms of ?3?4*-, ?4?2*- and ?5*-nAChR, which have repeatedly been linked to human smoking behavior. We have also observed differential macroscopic and single-channel functional effects of lynx1 across nAChR isoforms, which provide ideal readouts for use in defining the sites at which lynx1/?3?4*-nAChR interactions occur. These findings led us to our underlying hypothesis: that allosteric prototoxin effects arise from (generally well- conserved) interactions with non-agonist-binding nAChR ?(-)- subunit interfaces, and that differential outcomes arise from the details of interactions at each prototoxin/nAChR interface. New Preliminary data also indicate that rostral-IPN (IPR) GABA neurons, with a well-defined role in somatic nicotine withdrawal, coexpress ?3?4*- and ?4?2*-nAChR, together with high levels of both ?5 subunit and lynx1 mRNA. They therefore represent an excellent, dependence-related, native system for studying with which nAChR population(s) ?5 subunits associate, and how lynx1 modulates these same nAChR populations in the IPN. We therefore are ideally placed to compare functional outcomes of lynx1 modulation across the same defined nAChR populations in native neurons and in vitro models, enhancing validation and interpretation of findings across these systems. We will pursue this opportunity by combining precise experimental data from a multidisciplinary experimental approach with sophisticated molecular dynamics modeling. This closely integrated research plan will allow us to establish for the first time a generalized framework to understand how prototoxin modulators produce functional outcomes across multiple nAChR subtypes and isoforms, in both native neurons and in vitro expression systems. It will also ensure maintenance of scientific rigor, rapidly refine our experimental designs, and produce key biological and mechanistic insights. In addition, regionally-restricted prototoxin expression may permit modulation of nAChR function to be restricted to particular brain regions or cell types. Prototoxin/nAChR interactions may therefore represent promising new drug targets. By probing the nature and sites of prototoxin/nAChR interactions, this study promises to remove critical barriers to progress in scientific and clinical work related to nicotine addiction, as well as other conditions affected by nicotinic receptor function.